WO2016090361A1

WO2016090361A1 - Polymers for inducing 3d spheroid formation of biological cells

Info

Publication number: WO2016090361A1
Application number: PCT/US2015/064271
Authority: WO
Inventors: John Solomon GARNER; Sarah Michelle SKIDMORE; Justin Charles HADAR; Burnsoo HAN; Kinam Park
Original assignee: Akina, Inc. (An Indiana (Us) Corp)
Priority date: 2014-12-05
Filing date: 2015-12-07
Publication date: 2016-06-09
Also published as: US20170362566A1

Abstract

The present invention provides the use of selected thermogelling polymers for the purpose of growing tumor spheroids. The invention provides a thermogelling platform comprising a synthetic polymer which, when seeded with cancer cells, induces the cells to grow into a natural spheroidal pattern forming a tumor spheroid. After accomplishing this in about 3-10 days, the gel washes away, leaving behind the spheroids.

Description

Polymers for Inducing 3D Spheroid Formation of Biological Ceils

FIELD OF INVENTION

The present invention relates to the application of synthetic therrnogeliing polymers to the growth of spheroidally- formed biological cells. While cells grown in a 2D monolayer or other format lack physiological relevancy, spheroids are more representative of tumor formation as they exhibit increased cell survival, relevant morphology, and hypoxic core which is seen in native tumors but not in 2D or other tumor models. In order to assay potential treatments and for other research applications, there is a need for a 3D cell model, such as a spheroid, which accurately represents in-vivo conditions. This invention allows for the convenient growth of cell spheroids or other three-dimensional structures by co-incubation.

BACKGROUND OF THE INVENTION

Cells are cultured for a variety of reasons, including drug screening in cancer research , tissue engineering, tumor modeling, gene function analysis, and celt-cell interaction analysis (R. Z. Lin et a!., "Recent advances in three-dimensional multicellular spheroid culture for

biomedical research ". Biotechnology Journal 3 (2008) pp. 1 172- 1 184). Two-dimensional (2D) cell culture was initially developed in 1 908 (Harrison, R.G. et al. "Observations of the living developing nerve fiber". Anat. Rec. i, ( 1907) p. 3 16-128). While 2D cell culture has enjoyed a long history of use in the laboratory, it has been noted by researchers that ceils cultured in a three dimensional (3D) environment (spheroid) provide more clinically relevant data than ceils cultured in a monolayer. When cells are grown into a spheroid they are able to have cell-cell interactions as well as cell-extracellular matrix (ECM) interactions. These interactions more closely mimic the in vivo environment and can lead to more accurate data generation, particularly when drug screening tests are performed (S. Bresiin, et.al. "Three-dimensional cell culture: the missing link in drug discovery", Drug Discovery Today 18 (2013) pp. 240-249).

Spheroids have been used by researchers to study multicellular resistance to drugs (Desoize. B. et al.,"Ce/7 culture as spheroids: an approach to multicellular resistance.^''

Anticancer Res. 18 ( 1998), pp. 4147-4158 and Djordjevic, B., Lange, C.S., "Measurement of sensitivity to adriamycin in hybrid spheroids. " Cancer Invest. 9 ( 1991 ), pp. 505-512) . Spheroids are utilized in tumor modeling to determine signaling pathways and cell interactions to provide insight into drug treatment options, as well as drag development (Dardousis, K. et al.,

"Identification of differeniiaUy expressed genes involved in the formation of multicellular l mor spheroids by HT-29 colon carcinoma cells " Mol. Ther. (2007) 15, pp. 94-102; Ghosh, S. et at, "Use of multicellular tumor spheroids to dissect endothelial cell-tumor ceil interactions: a ro!e for T-cadherin in tumor angiogenesis". FEBS Lett. 581 (2007), pp. 4523-4528; Feder-Mengus, et al., "Multiple mechanisms underlie defective recognition of melanoma ceils cultured in three- dimensional architectures by antigen-specific cytotoxic T lymphocytes " Br. J. Cancer 96 (2007), pp. 1072-1082; and Durand, R.E., "Slow penetration of anthracyclines into spheroids and tumors: a therapeutic advantage?" Cancer Chemother. Pharmacol. 26 ( 1990), pp. 198-204). Spheroids are used extensively in drug screening platforms (Bartholoma, P., et ai., "Ά more aggressive breast cancer spheroid model coupled to an electronic capillary sensor system for a high-content screening of cytotoxic agents in cancer therapy: 3-dimensionat in vitro tumor spheroids as a screening model " J. Biomol. Screen. 10 (2005), pp. 705-714.; Friedrich, J., et ai., "Spheroid-based drug screen: considerations and practical approach." Nature Protoc. 4 (2009), pp. 309-324.; Kunz-Schughart LA, et al., ''The use of 3-D cultures for high-throughput screening: the multicellular spheroid model." J Biomol Screen 9 (2004) pp. 273-285.).

Spheroids have provided a useful alternative to mouse models in cancer research (Dubessy, C, et ai., "Spheroids in radiobiology and phoiodynamic therapy." Crit. Rev. Oncol. Hematoi. 36 (2000), pp. 179-192). Spheroids are a possible material for use in tissue engineering to reconstruct organs and whole tissues (Lin RZ, et ai., "Magnetic reconstruction of three- dimensional tissues from multicellular spheroids. " Tissue Eng Part C Methods 14 (2008) pp. 197-205; Marga F, et al., "Developmental biology and tissue engineering." Birth Defects Res C Embryo Today 81 (2007) pp. 320-328; Steer DL, Nigam S . "Developmental, approaches to kidney tissue engineering. " Am j Physiol Renal Physiol 286 (2004) pp. F 3 -F7).

Developing a 3D culture method has not always been a simple and inexpensive process. Currently, there are several methods to induce ceils to form a spheroid. One method is known as the "forced-floating" method. The surface upon which the cells grow is modified in a way thai the ceils are unable to attach to the surface. The cells are then forced to float, which induces cell-cell interactions leading to spheroid formation. (Ivascu, A. and Kubbies, M. "Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis." J . Biomol. Screen. 1 1 (2006), pp. 922-932; Friedrich, j. ei al. ¹¹ 'Spheroid-based drug screen:

considerations and practical approach. " Nat. Proioc. 4 (2009), pp. 309-324; Li, Q. et ai. "3D models of epilhelial- esenc ymal transition in breast cancer metastasis, " J. Biomol. Screen. 16 (201 1 ), p. 141-154). The pre-coating of the plates needed to develop spheroids using the forced- floating method can be time-consuming and labor intensive. Pre-coated plates are available to purchase from suppliers such as Sumitomo Bakeiite

(https://www.sumibe.co.jp/englishyproduct/s-bio/cell-culture/primesurfa

EMD Millipore (http://www.emdjmillipore.com/US/eri/l ife-science-research/cell-culture- systems/cell-growth/aEGb.qB.Cn4AAAE_kBd3.Mm_,nav) and Happy Ceil (http://www.happ>'- celi.com/happy-shop/low-attachment- lates/). However, purchasing pre-coated plates adds additional cost to the researcher's projects.

Another method of inducing spheroid formation is called the "hanging drop" method. In this method, a ceil suspension is seeded on a plate, which is then inverted and incubated. The cell suspension relies on surface tension to adhere to the plate while inverted. The ceils form spheroids after sinking to the bottom of the drop upon inversion and eu!turing. (Keim, J.M. et al . "Method for generation of homogeneous multicellular tumor spheroids applicable io a wide variety of cell types. ^'' Biotechnol. Bioeng. 83 (2003), pp. 173-1 80). While this method produces uniform spheroids, media change is very difficult and the volume of the drop containing ceils is limited (Kurosawa, H. "Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. " J. Biosci. Bioeng. 103 (2007), pp. 389-398). "Hanging drop" plates are commercially available from several suppliers including 3D Biomatrix

(www.3dbiomatrix.com) and InSphero fh ttp ://ww w. insphero . com") .

Yet another method for producing cell spheroids is an agitation-based procedure. In this method, either the ceils are stirred (Kim, J.B. "Three-dimensional tissue culture models in cancer biology. " Semin. Cancer Biol. 15 (2005), pp. 365-377) or the container the ceils are in is rotated (Barrila, J. et al. "Organotypic 3D cell culture models: using the rotating wall vessel io study hosl-pathogen interactions "^'' Nat. Rev. Microbiol . 8 (2010), pp. 791-801). The rotation of the cells promotes cell-cell interactions and inhibits cell adherence to the container wal ls. Some drawbacks of this method include large volume of media required, possible detrimental effect on cell physiology from rotation and limited control over spheroid size (Lin, R.Z. and Chang, H.Y. "Recent advances in three-dimensional multicellular spheroid culture for biomedical research " Biotechnology Journal, 3 (2008), pp. 1 1 72—1 184). Several companies have commercial agitators available, including Wheaton (http://www. wheatonsci.com), Corning ( http ://www.corni n g . com) and Synihecon (http://wwvv.svnthecon.com).

Another method for producing tumor spheroids involves the use of a matrix for spheroid development. The ceils are either seeded on, or in, a matrix. This matrix is a commercially purchased extra-cellular matrix that consists of biological material simi lar to what the ceil would be exposed to in-νίνο. The cost of purchasing this matrix may be prohibitive to most researchers and the composition of the matrix is not always consistent from batch to batch since it is a biological product. (Sodunke, T.R. et al. "Mrcropatte ns of Malrigel for three-dimensional epithelial cultures." Biomaterials 28 (2007), pp. 4006-401 6). Several ECM options are commercially available from companies such as BD Biosciences

(http://www.bdbiosciences.com/ca/cellculUire/rnimetic/indexjsp), Amsbio

(http:/7www.amsbio.com/animal-free-chemicali -defined-coilagen-laminin-nbronectin- vitronectin-ECM.aspx), Sigma-Aldrich

(http://www.sigmaaldrich.corn/catalog/product/sigma e 1270?lang=en&region=US), Miliipore (http://www.emdmillipore.com/US/en/product^^

C778 12) and Invitrogen (http://www.lifetecrmologies.corn/us/en/home/life-science/cell- culture/3d-cell-culture.html?cid=fl-3d-cellculture).

An additional mediod for producing spheroids involves the use of prefabricated scaffolds. Cells are seeded into the scaffold, where they attach to the fibers of the scaffold, and then grow into 3D structures. (Souria, A. et al. "Three-dimensional type I collagen gel system containing MG-63 osSeoblasSs-Uke cells as a model for studying local hone reaction caused by metastatic cancer cells,'^'' Anticancer Res. 16 ( 1996), pp. 2773-2780). Some difficulties with this method include the mechanical strength of the scaffold, cost and inability to remove the cells easily from the scaffold. Various scaffold types are commercially available from companies such as 3 D Biomatrix, Sigma-Aldrich, Solohill (ht^://www.pall.corn/main/biopharrnaceiiticals/solohill- microcarriers.page), and Amsbio (h ttp : //www.amsb io.com).

As discussed above, the various methods of ceil spheroid formation present numerous challenges to the researcher. The present invention eliminates these challenges with a novel platform for spheroid growth. This method is simple, inexpensive, and does not require extra equipment for spheroid development. SUMMARY OF THE INVENTION

The present invention provides the use of selected thermogelling polymers for the purpose of growing biological cells as spheroids. While the invention encompasses all biological cell types, tumor ceils are preferred. For example, the invention provides a thermogelling platform comprising a synthetic polymer which, when seeded with cancer ceils, induces the cells to grow into a natural spheroidal pattern forming a tumor spheroid. After accomplishing this in about 3- 10 days, the gel washes away, leaving behind the spheroids.

In a i ^si aspect, the present invention provides a platform for inducing 3D spheroid formation of biological ceils, comprising: a thermogelling polymer without amino acids.

In a l^si embodiment, the thermogelling polymer is chemically synthetic or semi -synthetic.

In a 2^nd embodiment, the thennogelling polymer is a stearate-modified methyl cellulose.

In a 3^rd embodiment, the thennogelling polymer is poloxamer 407 linked by polyurethane linkages.

In a preferred embodiment, the polyurethane linkages are generated utili2ing

hexamefhyidiisocyanate.

In another preferred embodiment, the polyurethane linkages are generated utilizing methylene diphenyldiisocyanate.

In another preferred embodiment, the polyurethane linkages are generated utilizing toluene diisocyanate.

in a 4^ϋ! embodiment, the thermogelling polymer is a hydrophobica!ly-modified cellulose derivative.

In a 5^Ul embodiment, the thermogelling polymer is a cellulose derivative modified by chemical conjugation of stearic acid units.

In a 6^ϋϊ embodiment, the thermogelling polymer is a cellulose derivative modified by chemical conjugation of methyl units.

In a 7^th embodiment, the thermogelling polymer is a cellulose derivative modified by chemical conjugation of hydroxypropyi units.

In an 8^th embodiment, the thermogelling polymer is a cellulose derivative modified by chemical conjugation of ethyl units. In a 9 embodiment, the thermogelling polymer is a cellulose derivative modified by chemical conjugation of propyl units.

In a 10 embodiment, the thermogelling polymer is a cellulose derivative modified by chemical conjugation of hydro xyefhyl units.

In an 1 1 ^ϋ1 embodiment, the thermogelling polymer is a cellulose derivative modified by chemical conjugation of carboxymethyl units.

In a 12 embodiment, the platform further comprises a cell growdi medium in die range of about 3 to about 30% w/v.

in a preferred embodiment, the cell growth medium is in the range of about 5 to about 20% w/v.

In a 13^ih embodiment, the biological cells are tumor cells.

In a 14 embodiment, the biological cells are benign tumor cells.

In a 15^th embodiment, the biological cells are malignant tumor cells.

In a \ 6^>h embodiment, the biological cells are cancer ceils.

In a 17^ih embodiment, the biological cells are breast cancer ceils.

In an 18^u> embodiment, the spheroids have diameters of about 10 μ^ηι to about 1000 urn.

In a 19^ih embodiment, the spheroids have diameters of about 10 μηι to about 500 μπι.

In a 20^ώ embodiment, the spheroids have diameters of about 20 jxm to about 200 μηι.

In a 2"^d aspect the present invention provides a method for inducing 3D spheroid formation of biological cells on a thermogelling polymer without amino acids, comprising the step of: combining the thermogelling polymer with a growth medium and the biological ceils.

In a i ^si embodiment, the thermogelling polymer is chemically synthetic or semi-synthetic.

In a 2"^d embodiment, the thermogelling polymer is a stearate-modified methyl cellulose.

In a 3^rd embodiment, the thermogelling polymer is poloxamer 407 linked by polyurethane linkages.

In a preferred embodiment, the polyurethane linkages are generated utilizing

hexamethyidiisocyanate.

i another preferred embodiment, the polyurethane linkages are generated utilizing toluene diisocyanate. in a 4 ¹ embodiment, d e thermogelling polymer is a hydrophobically-modif ed cellulose derivative.

In a 5^!ii embodi ment, the thermogelling polymer is a cellulose derivative modified by chemical conjugation of stearic acid units.

In a 6^ϋί embodiment, the thermogelling polymer is a cellulose derivative modified by chemical conjugation of methyl units.

In a 7^th embodiment, the thermogelling polymer is a cellulose derivative modified by chemical conjugation of hydroxypropyl units.

In an 8^ώ embodiment, the thermogelling polymer is is a cellulose derivative modified by chemical conjugation of ethyl units.

In a 9^Ui embodiment, the thermogelling polymer is a cellulose derivative modified by chemical conjugation of propyl units.

In a 10^th embodiment, the diermogell ng polymer is a cellulose derivative modified by chemical conjugation of hydroxyethyl units.

In an 1 1^ϋϊ embodiment, the diermogelling polymer is a cellulose derivative modified by chemical conjugation of carboxymethy! units.

In a 1 2^th embodiment, the ceil growth medium is in the range of about 1 to about 30% w/v.

in a 1 3^th embodiment, the biological cel ls are tumor cells.

In a 14^α' embodiment, the biological cells are benign tumor cells.

In a 15^th embodiment, the biological cells are malignant tumor cells.

In a 16^ih embodiment, the biological cells are cancer ceils.

In a 17^ embodiment, the biological cells are breast cancer ceils.

In an 18^ϋ! embodiment, the spheroids have diameters of about 10 μπι to about 1000 μηι. in a 19^th embodiment, the spheroids have diameters of about 10 μπι to about 500 μ^ηι.

In a 20^th embodiment, the spheroids have diameters of about 20 μηι to about 200 pm.

In a 21^st embodiment, the method further comprises the step of: incubating the biological cells with the thermogelling polymer and growth medium at appropriate conditions to support biological growth. In a 22" embodiment, the method further comprises the step of: harvesting the 3D spheroid formations of biological cells from the ihemioge!ling polymer and growth medium.

It will be appreciated that all allowable combinations of the above aspects/embodiments, as well as other aspects/embodiments described elsewhere herein, are contemplated as fiirther aspects/embodiment of the invention.

BRIEF DESCRIPTION OF TEE DRAWINGS

It will be appreciated that the following Figure relate to a particular embodiment of the invention discussed below. The Figure is not intended to limit the scope of the invention.

FIGURE 1 : MCF-7 cells growing in A025 after one day of incubationG.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein,

The term "biological cell," unless modified, refers to any biological cell. Preferred biological cells include tumor and cancer cells.

The term "synthetic polymer" refers to a chemical substance, not derived from natural origins, consisting of repeating units linked together with covaient bonds.

The term "semi-synthetic polymer" refers to a polymer prepared by chemical synthesis starting from natural materials.

The term "biocompatible" refers to a material property which allows for the growth and/or proliferation of biological cells in its presence.

The term "growth medium" refers to a liquid or gel which supports the growth of microorganisms or biological cells. For example, in some embodiments, the medium contains water and nutrients.

The term "thermogel" refers to any polymeric material that, upon dissolution in water, possesses the property to change solution rheological properties upon change in solution temperature.

The invention relates to the use of a selected series of synthetic thermogel polymers for the purpose of growing cancer cells into spheroids. When dissolved in water at a gellable concentration (e.g., 1 % to 50% w/v, preferably 5% w/v to 20% w/v at 5C), the thermogel should have a viscosity of less than 100 Pa.s, preferably less than 50 Pa.s, more preferably less than 1 0 Pa.s. in fact, as there is no lower limit, a viscosity of 0 is optimai. Unlike other growth media, these synthetic polymers are not derived from biological sources, and as such are lower in cost and higher in reproducibility than other materials used for this application. The synthetic pathways for these thermogeis are described in examples below.

Abbreviations

The following abbreviations are used throughout this document:

discrete spheroid growth {approximately 60- 95% aggregated)

Practically complete aggregated growth / sheet

4

like ceil structures (>95% aggregated)

/. Syntheses:

Example I AO 14 PolviN-isopropylacrylamide-co-acrylic acid) This copolymer is synthesized by charging into a 1000 ml 1 neck round-boltom-fiask 75 grams of NIPAM, 5 ml of A Ac, 240 ml of DW, 240 ml ACN, 0.4 ml TEMED, and 0.4 g APS. The solution is sparged with nitrogen gas and gentle warming at 60 °C to initiate polymerization. Post reaction, the polymer solution is dissolved in a mixture of DCMACE ( 1 : 1 v:v), passed through a filter, and precipitated in hexet. Obtained solid is dried under deep vacuum at room temperature.

Example 2: AO20 Pd!vfF127~ureihane) (~2 units)

Into a i L RBF is put 1 00 g of F3 27 plus 4 grams mPEG (5000 Da). This is dried at room temperature under deep vacuum to remove surface water. These are then dissolved in anhydrous DCM and 10 mi of 10% w/v Sn(Oct)/toluene is added along with 1 .35 mi of HMDL The solution is sealed and heated at 60 °C overnight under reflux with a desiccant trap. Polymer is dissolved in dichioromethane, passed through a fi lter, and precipitated in hexet. Obtained solid is dried under deep vacuum at room temperature.

15 Stearic acid modified methvlcellul

Into a 1000 ml flask 3 1 .4 g of methylcelluiose (Mn 14,000, 3 .6- 1.9 mol methoxy per moi cellulose, 27.5-3 1 .5% wt% methoxy, 53-59 dynes/cm surface tension (25C, 0.05%, 15 cP, gei point 50-55 °C) is added along with anhydrous ACN plus 2.2 mi triethyiamine. This is stirred and heated to 90 °C to dissolve. To this solution 2.52 g of stearyloyl chloride is added along with 100 mi of D SO, and the solution is stirred at room temperature for 3 days. Subsequently, this solution has its volume reduced to roughly 1/2 - 3/4 of current volume under vacuum and then is precipitated in ethanol. Obtained solid is washed with ethanol and dried under deep vacuum at room temperature.

Example 4: AQ26 PolyfPoIoxamer 407-methvier¾edipher¾vitliisocsariate into a round-bottom-flask is placed 100 g F127, 0.8 g mPEG (5000 Da) and heated to 1 25 °C while under deep vacuum to dehydrate for 1 hour. Subsequently, 3 g of MD1 is melted at 80 °C and combined with pre-heaied F 127/mPEG 5000 mixture. This is stirred rapidly, then allowed to coot to room temperature. After cooling, polymer is dissolved in DCM, filtered, and precipitated in hexet. Obtained solid is dried under deep vacuum at room temperature.

Example 5: AQ31 Poiyiv½YlcaprQlactam j

Into a 1000 mL, 2 neck round bottom flask 2.5 mi of 4% (w/v) AIBN/DMSO is added along with 50 g vinyicaproiactam. This is then dissolved with -300-500 mi aceton itrile. The solution is sparged for 30 minutes to remove oxygen, sealed off, and heated to 70°C overnight to react. Subsequently, this polymer is precipitated i n hexet, and the obtained solids are dried under deep vacuum at room temperature.

//. Characterization:

The hydrogei Examples described above are characterized by the following methods: Rheological Characterization

Rheology was performed on a TA instruments AR550 equipped with a temperature controlled peltier plate. The geometry utilized is a 60 mm 2° cone with a 62 μπι truncated tip. The peltier plate has a heatsink provided by a Neslab RTE 10 refrigerated circulator set to pump solution through the peltier plate at 23°C. The air bearing is supported by 35psi of compressed air. The instrument is operated by an Inspiron 530s Dell computer utilizing AR instrument control software. Data analysis is provided on same computer using TA universal data analysis software. Prior to initiating runs the instrument geometry was mapped utilizing AR instrument control software using settings of standard mapping speed at 3 iterations. Platen was set to an initial temperature of 5 °C. Subsequently the gap was zero using software provided feature and gap was set to 100pm for sample loading. Each polymer was dissolved in cold water as detailed in the examples and the generated solution was stored in an insulated container along with an icepack prior to testing.

Each sample (2 ml) was injected into the gap set to 100 μπι and the gap was reset to 62 μτη for the run. For the run, each sample was initially equil ibrated at 5 °C for 1 min prior to testing. At 5 °C, an applied shear rate of 0. 3 sec^"! was applied and the sample and the resultant viscosity was sampled at 5 second intervals. After this, the sample temperature was increased stepwise from 5 °C to 45 °C at 2.5 °C increments with 3 minutes of equilibration time at each increment. At each increment, 0. 1% strain was applied with an oscillating frequency of 6.283 rad/sec with a conditioning time of 3 seconds and a sampling time of 3 seconds. After measuring

U at the final 45 °C temperature, the sample was returned to 5 °C. The cone was raised and the cone/platen cleaned off prior to injecting the next sample. The viscosity of each sample at 5 °C was calculated from the initial shear rate run by averaging the viscosity values of each rime interval. This value is listed in results section for each example.

Inherent Viscosity

For selected polymers in which gel permeation chromatography was not possible, inherent viscosity was performed in order to obtain molecular weight information. Each sample was prepared by dissolving at indicated concentration (typically 3 -2% w/v ( 1 -2 g dL)) in indicated solvent as detailed in each example. Each sample was dissolved at room temperature. Samples were tested in a Cannon mini-viscometer (C286) in a water bath set to the indicated temperature. The test result (in seconds) was multiplied by the manufacturer provided

viscometer constant (0.004039 cST/s) to obtain sample viscosity. The relative viscosity (V_re[) was obtained by dividing the sample viscosity by that of literature reported value for pure solvent. The inherent viscosity (IV) was obtained (in dL/g) by the following equation where "C" is the polymer concentration:

IV (dL/g) = in(V_re])/(C g/dL)

Testing was performed in triplicate and 3 sample results were averaged together for each example unless otherwise specified.

Gel Permeation Chromatography (GPC)

Select polymers were tested by GPC. Each sample was dissolved in 0.2um filtered chromatography grade dichloromethane (DCM) (Mailinckrodt, Chromasolv). After dissolution, the sample was passed through a 0.45 μηι PVDF filter to remove any particulates, and placed directly into a septa capped 2mi HPLC vial. Gel-permeation chromatography was performed using a Varian prostar system equipped with a model 2 10 isocratic pump, a model 4 10 auto- sampler, and a model 335 phoiodiode array (PDA) detector. Elution was done with I mL/min flow of DCM across three col umns in sequence containing a combination of sized phenogel and Resipore (Agilent, mixed bed) columns. Unless otherwise specified, absorbanee of the sample was taken at 237nm . Control was performed using Galaxie software package. Cal ibration of the system was performed using Agilent PS2 © polystyrene standard series "A" and "B" per MFG instructions. Calibration was performed in a "book -end" format with standards run before and after the sample runs and their retention times averaged to generate the calibration curve. Hydrogen-nuckar magnetic resonance (HNMR)

For select polymers, a portion of the purified solids was dissolved in an appropriate duecerated solvent (either deuterium oxide, Deuterated chloroform, or Deuterated methyl sulfoxide depending on solubility). The solution was placed in an HNMR glass tube and analyzed by HNMR at a minimum frequency of 300 MHz.

FTIR

Polymers which were soluble in dichloromethane were dissolved in DCM and cast coated onto salt plates. Polymers that were not soluble in dichloromethane were ground to a fine powder, mixed with potassium bromide and compressed into a pel let. The samples were measured using a Thermo Malison, Satellite FTIR, Model 96GM0017 with standard sample holder. This instrument was operated and data analyzed utilizing WinFirst software. Each run was performed by doing 35 scans from 400 cm^"' to 4000 cm^"1 in transmittance mode at a resolution of 4.0 and a gain of 1 . The mirror, processing, and signal settings were all at factory default. Prior to loading samples a background scan was collected with the sample chamber empty. After a background scan was collected each sample was placed in the sample chamber in the laser path and scanned. Cell Growth

Hydrogels were weighed into scintillation vials and sterilized using Anproiene AN74i ethylene oxide gas sterilizer for 12 hour cycle per manufacturer's instructions. Hydrogels were then dissolved in cell growth media which consisted of DMEM/F 12 + Glutamax ¹ basal media supplemented with 5%v/v fetal bovine serum, penicillin ( 100 units/mL), and streptomycin (100 [ig/mL). Appropriate amount of growth media was added to give desired %w/v concentration for each hydrogel. After adding cell growth media hydrogels were allowed to fully dissolve at refrigerated conditions (approximately 2-8 °C) for -48 hours.

MCF-7 ceils were cultured in 75 cm² flasks until ~70-90% confluence was reached. Cel ls were prepared by first removing media from culture flask, washing with DPBS (Dulbecco's Phosphate Buffered Saline) and then adding 0.025% trypsin in EDTA solution and allowing 3-5 minutes of incubation at 37 °C/5% C0₂ to detach ceils from the surface of the flask. Fresh media was then added to flask to neutralize trypsin activity and re-suspend cells. This cell suspension was then transferred to a 15 mL conical bottom tube and centrifuged at -200 g for three minutes to produce a cell pellet in the bottom of the tube. Media was then removed carefully as to riot disturb the ceil pellet. At this point the cell pellet was re-suspended in fresh media or cold hydrogels dissolved in media.

Hydrogels were pipetted into flat bottom polystyrene multi-well tissue culture plates with low evaporation lid. These were then placed in VWR Symphony 3405 air jacketed incubator at 37 °C/5% CC for 90 minutes to firm hydrogels. Hydrogels were then seeded with MCF-7 cells by pipetting cel ls suspended in media on top of gels and rocking plates back and forth to disperse cell suspension. After seeding with MCF-7 cells, multi-well plates were placed in incubator at 37 °C/5% CO . Experiments were also performed by suspending ceil pellets in cold

hydrogei/ceil media mixtures using AO20 and A025. These mixtures were then pipetted into multi-well plates and placed in incubator at 37 °C/5% CO?.

Procedures described above including dissolving hydrogels in growth media and seeding cells on hydrogels were carried out under aseptic conditions using Esco class 11 type A2 biological safety cabinet. After a period of incubation time ranging from twenty-four hours to seven days, photos were taken using Aniscope FN300i-FL phase contrast microscope. During the time ceils were incubated media was replaced approximately every 48-72 hours or as color change of media indicated drop in pH.

//. Cell Growth Exam les:

After syndtesis, the Example thermogei was characterized as described above with the following results:

Rheologieal

The viscosity of Example i for a 1 % (w/v) solutio was found to be 0.07059 Pa.s in water at 5 °C. The rheological G' for example 1 % in water hit a maximum of 0.1 Pa at 45°C. The gelation properties of AO 1 4 were separately found to be dependent on pH with a favoring of gelation at low pH values.

HNMR

The ITNMR spectra of Example 1 indicates peaks at following locations and intensities in following format location ppm (intensity, description): 1 .0 ppm ( 1 00.00, broad), 1 .5 pprn(34.76, broad), 2 ppm ( 1 8. 1 1 , broad), 2.7 ppm (0. 19, single), 3.6 ppm (0J I , single), 3.8 ppm ( 1 6.45, broad). 7.7 ppm (2. 14, broad). These results are consistent with the indicated polymer indicating successful synthesis. FTIR

The FTIR spectra indicated a broad peak at 3500-3300 cm^"1, a sharp triplet peak at 2900- 2700 cm^"1, strong absorption peaks at 1700 cm^{" 5}, 1500 cm^'1, as well as weaker peaks at 1400 cm^" 1300 cm^"', and a broad peak at 1200- 1 100 cm^"1. These results are consistent with the indicated polymer indicating successful synthesis.

Cell Growth

Cell growth in AO 14 1 % (w/v) dissolved in cell growth media resulted in typical two dimensional cell morphology with a flat, trapezoidal shape. Cells adhered to surface of wells much like typical growth in culture flasks. This Example shows that not any thermogei can function to generate 3D spheroidal pattern.

CCD-1068SK Fibroblasts: No* tested,

HEP G2 Hepatic Cells: Not tested,

VERO African Green Monkey Kidney Cells: Not tested.

Example 2. Polyj Pieronic F127-urethane (AO20)

After synthesis, the Example thermogei was characterized as described above with the following results:

RheologicaS

For rheo!ogicai testing a 30% w/v solution was generated in cold water. This solution was first tested for viscosity which was found to be 0.1018Pa.s at 5 °C. Upon temperature ramping the maximum gel strength obtained was a G^* of 4000 at 45 °C with onset at 27.5 °C. The G' at 37°C was 1500 Pa.

HNMR

HNMR spectra was collected from polymer dissolved in Deuterated water. The HNMR spectra of Example 2 indicates peaks at following locations and intensities in following format "location" ppm (intensity, description): 1.2 ppm (0.75, broad), 1.7 ppm ( 16.03, broad), 1 .9 ppm ( 100.00. broad), 2.1 ppm ( 1.07, broad). 2.4 ppm (0.46, broad). These results are consistent with the indicated polymer indicating successful synthesis.

FTIR

The FTIR spectra indicated a broad peak at 3500-3300 cm⁴, a strong peak at 3000-2700 cm^"1, weak absorption peaks at 2600 cm^"1, 1 00 cm^"1 , 1700 cm^"! , 1500 cm^"', 1400 cm^"1, 1300cm- i , and 900 cm^"! as well as a very strong broad peak at 1200- 1000 cm^'' . These results are consistent with the indicated polymer indicating successful synthesis.

Cell Growth

MCF-7 Breast Cancer:

Cell growth in AO20 10% (w/v) dissolved in cell growth media resulted in three dimensional ceil morphology. Ceils grew spheroids suspended in hydrogel unlike typical growth in culture flasks. Spheroidal colonies of cells are suspended throughout the hydrogel at varying depths from surface of multi-well culture plate. Cellular growth was observed four days after seeding ceils on AO20 hydrogel that has been thermally gelled before adding ceil suspension on top. Cell growth was also observed seven days after cells suspended in cold hydrogel

Spheroids reach a larger size when seeded on warmed hydrogel versus those suspended in cold hydrogel. Spheroids in warm hydrogel range in size from 60- 120 μηι with most being—100 μη . Spheroids grown by mixing in initially liquid hydrogel range in size from 20-60 μηι with a fairly even size distribution. Level of aggregation 0 after four days of growth.

CCD-1068SK Fibroblasts:

Ceil growth in AO20 10% (w/v) dissolved in ceil growth media resulted in three dimensional cell morphology. Cells grew spheroids suspended in hydrogel unlike typical growth in culture flasks. Spheroidal colonies of cells are suspended throughout the hydrogel at varying depths from surface of multi-well culture plate. Ceils showed spheroid formation when seeding on AO20 hydrogel that has been thermally gelled before adding ceil suspension on top as well as cells suspended in cold hydrogel. Spheroids ranged in size from 40-180μηι with most being ~80μπι. Level of aggregation 0 after four days of growth.

HEP G2 Hepatic Cells:

Ceil growth in AO20 10% (w/v) dissolved in ceil growth media resulted in three dimensional cell morphology. Cells grew spheroids suspended in hydrogel unlike typical growth in culture flasks. Spheroidal colonies of cells are suspended throughout the hydrogel at varying depths from surface of multi-well culture plate. Cells showed spheroid formation when seeding on AO20 hydrogel that has been thermally gelled before adding cell suspension on top as well as cells suspended in cold hydrogel. Spheroids ranged in size from 60-160μηι with most being ~80μιη. Level of aggregation 2 after four days of growth.

VERO African Green Monkey Kidney Cells: Ceil growth in AO20 10% (w/v) dissolved in cell growth media resulted in three dimensional cell morphology. Ceils grew spheroids suspended in hydrogel unlike typical growth in culture flasks. Spheroidal colonies of cells are suspended throughout the hydrogel at varying depths from surface of multi-well culture plate. Cells showed spheroid formation when seeding on AO20 hydrogel that has been thermally gelled before adding cell suspension on top as well as cells suspended in cold hydrogel. Spheroids ranged in si2e from 40-200μηι with even size distribution. Two dimensional attached growth also occurred simultaneously in some wells. Level of aggregation 1 after four da s of growth.

After synthesis, the Example thermogel was characterized as described above with the following results:

Rheologkal

Example 3 was dissolved 5% w/v in distilled water and tested by rheology. The viscosity of the 5% w/v solution at 5C was 0. ! 742 Pa.s. The maximum G^! reached was 80Pa at 45°C with onset at 35°C.

HNMR

The HNMR spectra of Example 3 indicates peaks at following locations and intensities in following format "location" ppm (intensity, description): 1.0 ppm (0.58, doublet), 2.7 ppm (44.30, broad), 3.0 ppm (8.70, broad), 3.3-4.0 ppm ( 100.00, broad), 4.7 ppm (9.33, broad). These results are consistent with the indicated polymer indicating successful synthesis.

FT! R

The FTIR spectra indicated a broad peak at 3600-3300 cm^" , a weak peak at 2900 cm^" , a strong peak at 1200- 1000 cm^"', and a weak peak at 900 cm^"' . These results are consistent with the indicated polymer indicating successful synthesis.

Cell Growth

MCF-7 Breast Cancer:

Ceil growth in A025 5% w/v dissolved in cell growth media resulted in three

dimensional ceil morphology. Cells grew spheroids suspended in hydrogel unlike typical growth in culture flasks. Spheroidal colonies of cells are suspended throughout the hydrogel at varying depths from surface of multi-well culture plate. Cell growth -24 hours after seeding cells on A025 hydrogel that has been thermally gelled before adding cell suspension on top was observed. Cell growth seven days after ceils suspended in cold hydrogei was also observed. Spheroids reach a larger size when seeded on warmed hydrogei versus those suspended in co!d hydrogei. Spheroids grown on warmed hydrogei range in size from 30- 1 10 μπι with most being larger than 50 μηι. Spheroids grown by mixing in liquid hydrogei range in size from 20-60 μηι with a fairly even size distribution. The image in Figure 1 shows MCF-7 cells as described in Example 3 incubated for approximately 30 hours. The red arrow indicates an example of ceils which have morphology indicating spheroidal growth. This is indicated by the overall spheroid morphology with fused cells that do not show distinguishable individual ceil boundaries, i.e., without distinctive cellular membrane at cell-ceil interfaces. Level of aggregation 0 after one day of growth and subsequently at four days of growth,

CCD-1068SK Fibroblasts:

Ceil growth in A025 5% w/v dissolved in cell growth media resulted in three

dimensional ceil morphology. Ceils grew spheroids suspended in hydrogei unlike typical growth in culture flasks. Spheroidal colonies of cells are suspended throughout the hydrogei at varying depths from surface of mufti-well culture plate. Spheroids reach a larger size when seeded on warmed hydrogei versus those suspended in cold hydrogei. For seeding on warmed hydrogei spheroids ranged in size from 40- 180μτη with most being ~- I ΟΟμη , Seeding by suspending cells in cold hydrogei resulted in spheroids initially 20-60 μιτι but after extended incubation times (i.e. greater than 10 days) with media replacement spheroids increased in si2e to ~10Ο-32Ομηι, Level of aggregation 1 after four days of growth.

HEP G2 Hepatic Cells:

Ceil growth in A025 5% w/v dissolved in cell growth media resulted in three

dimensional cell morphology. Ceils grew spheroids suspended in hydrogei unlike typical growth in culture flasks. Spheroidal colonies of cells are suspended throughout the hydrogei at varying depths from surface of multi-well culture plate. Spheroids ranged in size from ό0- 140μτη with most being ~80μηι. Additionally some irregular ceil growth occurred in three dimensional sheets of cells suspended in gel. Level of aggregation 3 after five days of growth.

VERO African Green Monkey Kidney Cells;

Ceil growth in A025 5% w/v dissolved in cell growth media resulted in three

dimensional cell morphology. Cells grew spheroids suspended in hydrogei unlike typical growth in culture flasks. Spheroidal colonies of cells are suspended throughout the hydrogei at varying depths from surface of multi-well culture plate. Spheroids range in size from 20- 120 μπ with a most being ~80 μηι. Level of aggregation 2 after one day of growth and at four days of growth.

Example 4, PoIyfPoloxamer 407-methylened¾pheiividi¾socianate) (AQ26) After synthesis, the Example thermogel was characterized as described above with the following results:

Rheological

The polymer was dissolved as a 5% w/v solution in cold water and tested by theology. The viscosity of this solution at 5 °C was found to be 0.1801 Pa.s. The highest measured G' was 440 Pa with gelation onset around 15 °C. The G' at 37 ^aC was 130 Pa.

HNMR

The HNMR spectra of Example 4 in CDCB indicates peaks at following locations and intensities in following format "location ppm (intensify, description): 0.9ppm (9.33, broad), 1.0 ppm ( 3.05, sharp), 1.2 ppm ( 18.80, broad), 1 .3 ppm (9.47, broad), 1.7 ppm (5.86, multiple) 3.3 ppm (6.15, broad), 3.5 ppm ( 100.00, broad), 3.7 ppm (0.69, sharp), 3.9 ppm ( 1.82, broad), 4.3 ppm (0.33, broad), 5.2 ppm (0.06, sharp), 6.0 ppm (0.12, triplet), 7.0 ppm (0.81 , broad), and 7.4 ppm (0.51 , broad). These results are consistent with the indicated polymer indicating successful synthesis.

TIR

The FTIR spectra indicated strong peaks at 3000-2700 cm^{' 1} and 1200 cm^"1 and medium peaks at 1 50 cm^"5, 1350 cm^"1, 1300 cm^{" 5}, 1250 cm^{" 5}, 900 cm^{" 5} and 800 cm^{" 1}. These results are consistent with the indicated polymer indicating successful synthesis.

Cell Growth

MCF-7 Breast Cancer:

Ceil growth in A026 5% w/v dissolved in cell growth media initially resulted in two dimensional cell morphology with a flat, trapezoidal shape but with somewhat rounded edges. Cells adhered to surface of wells much like typical growth in culture flasks. After a period of 5 days the same well in multi-well culture plate shows that cells have detached from the surface and formed spherical shaped cell colonies. These spherical growths appear to be contained in a naiTow portion of the hydrogeis just above the surface of the multi-well culture plate. Some of the growths are irregular in shape being more oblong than spherical . They range in size from 20- 120 μηι with most being ~75

Level of aggregation 1 after five days of growth. CCD-1068S Fibroblasts:

Cell growth in A026 5% w/v dissolved in cell growth media resulted in three

dimensional cell morphology. Cells grew spheroids suspended in hydrogel unlike typical growth in culture flasks. Spheroidal colonies of cells are suspended throughout the hydrogel at varying depths from surface of multi-well culture plate. They range in size from 40- 120 μπι with most being -80 μηι. Level of aggregation 1 after four days of growth.

HEP G2 Hepatic Cells:

Ceil growth in A026 5% w/v dissolved in cell growth media resulted in three

dimensional ceil morphology. Ceils grew spheroids and thick sheets of cells suspended in hydrogel unlike typical growth in culture flasks. Spheroidal colonies of cells are suspended throughout the hydrogel at varying depths from surface of multi-well culture plate. Level of aggregation 3 after two days of growth.

VERO African Green Monkey Kidney Cells:

Ceil growth in A026 5% w/v dissolved in cell growth media resulted in three

dimensional cell morphology. Ceils grew spheroids suspended in hydrogel unlike t pical growth in culture flasks. Spheroidal colonies of cells are suspended throughout the hydrogel at varying depths from surface of multi-well culture plate. They range in size from 40- 340 μιη with most being ~80 μπη. Some surface growth also occurred. Level of aggregation 2 after four days growth.

Example 5. PoivCvinyleaprolaetarn^' (AQ3

After synthesis, the Example thermogel was characterized as described above with the following results.

R eoSogical

The example 5 polymer was dissolved as 20% w/v in cold distilled water. This was tested by rheology and the viscosity at 5°C was found to be 0.07835 Pa.s. The maximum G' obtained upon temperature ramp was 100,000 Pa at 45 °C with onset initiating at 30 °C The G' at 37 °C was 60,000 Pa.

HNMR

The HNMR spectra was collected in D₂0. The HNM R spectra of Example 5 indicates peaks at following locations and intensities in following format "location" ppm (intensity, description): 0.9ppm (3.33, split), 1 .2-2.0 ppm (58.40, broad), 2.0-2.75 ppm ( 17.56, broad), 2.7- 3.7 ppm ( 16.83, broad), 3.7-4.5 ppm (6.85, broad) 5.7 ppm (0.55, multiple). These results are consistent with the indicated polymer indicating successful synthesis.

FTIR

The FTIR spectra indicates strong peaks at 3500-3300 cm^"1 , 3000-2700 cm^"1, 1700 cm^"1 , 1400 cm^{" 1} and medium peaks at 2300 cm^"1, 1300- 1000 cm^{" 1} , 900 cm^"1, 800 cm^"1 , 700 cm^{" 1} and

600 cm^{" 1}. These results are consistent with the indicated polymer indicating successful synthesis.

Cell Growth

MCF-7 Breast Cancer:

Ceil growth in A031 20% (w/v) dissolved in ceil growth media initially resulted in typical two dimensional cell morphology with a flat, trapezoidal shape. Ceils adhered to surface of wells much like typical growth in culture flasks. Ceils would rapidly die and detach from surface and appear as sheets of ceils floating in media. Similar to Example 1 , this Example shows that only specific thermogelling polymers work.

CCD-1068SK Fibroblasts: Not tested.

HEP G2 Hepatic Cells: Not tested.

VERO African Green Monkey Kidney Cells: Not tested.

IV. Discussion

Of the polymers tested, there are two important properties in regards to the polymer having the capacity to serve as a spheroid-activating growth media.

First, the polymer must have the capacity to form into a thermogei at 37 °C which has suitable stiffness and viscosity to mimic the mechanical properties which normally encompass cancerous cells in vivo. Example 1 shows a weakly thermogelling polymer in which the cells simply grow in a traditional 2D format along the bottom of the well.

Second, the polymer must have suitable biocompatibility to allow for cell attachment and growth. As shown in Example 5, the polymer is not conducive to cell growth despite being a thermogei. Cellular extracts, e.g., Matrigei, or synthetic poly(amino acids) have been used as a matrix to grow 3D ceil spheroids, but this is the first time to show that synthetic and semisynthetic polymers which do not have amino acids display the property of promoting 3D ceil spheroid formation. These new synthetic and semi-synihetic thermogeliing polymers allow for growing tumor cells in a media which is conducive towards generating bio-relevant tumor morphology in a reproducible manner.

it will be appreciated that the Examples presented herein are meant to further describe the invention, and not to limit the scope thereof.

Claims

WE CLAIM:

3 . A platform for inducing 3D spheroid formation of biological ceils, comprising: a ihermogelling polymer without amino acids.

2. The platform for inducing 3D spheroid formation of biological cells of claim 1 , wherein the thermogeiling polymer without amino acids is a siearate-modified methyl cellulose.

3. The platform for inducing 3D spheroid formation of biological ceils of claim 1 , wherein the thermogeiling polymer without amino acids is poloxamer 407 linked by

poiyurethane linkages.

4. The platform for inducing 3D spheroid formation of biological ceils of claim 3, wherein the poiyurethane linkages are generated utilizing hexamethyldiisocyanate.

5. The platform for inducing 3D spheroid formation of biological ceils of claim 3 , wherein the poiyurethane l inkages are generated utilizing methylene diphenyldiisocyanate.

6. The platform for inducing 3D spheroid formation of biological ceils of claim 3, wherein the poiyurethane linkages are generated util zing toluene diisocyanate.

7. The platform for inducing 3D spheroid formation of biological ceils of claim 1 , wherein the thermogeiling polymer without amino acids is a cellulose derivative modified by chemical conjugation of stearic acid units.

8. The platform for inducing 3D spheroid formation of biological ceils of claim 1 , wherein die thermogeiling polymer without amino acids is a cellulose derivative modified by chemical conjugation of methyl units.

9. The platform for inducing 3D spheroid formation of biological ceils of claim 1 , wherein the thermogeiling polymer without amino acids is a cellulose derivative modified by chemical conjugation of hydroxypropyi units.

10. The plaiform for inducing 3D spheroid formation of biological cells of claim 1 , wherein the thermogeliing polymer without amino acids is a cellulose derivative modified by chemical conjugation of ethyl units.

1 1 . The platform for inducing 3D spheroid formation of biological cells of claim 1 , wherein the thermogeliing polymer without amino acids is a cellulose derivative modified by chemical conjugation of propyl units.

12. The platform for inducing 3D spheroid formation of biological ceils of claim 1 , wherein the thermogeliing polymer without amino acids is a cel lulose derivative modified by chemical conjugation of hydroxyeihyl units.

13. The platform for inducing 3D spheroid formation of biological cells of claim 1 , wherein the thermogeliing polymer without amino acids is a cellulose derivative modified by chemical conjugation of carboxymethyl units.

14. The platform for mducmg 3D spheroid formation of biological ceils of claim 1 , further comprising a cel l growth medium in the range of about 1 to about 30% w/v.

15. The platform for inducing 3D spheroid formation of biological ceils of claim 1 , wherein the ceil growth medium is in the range of about 5 to about 20% w/v.

16. The platform for inducing 3D spheroid formation of biological ceils of claim 1 , wherein the biological cells are tumor cells.

17. The platform for inducing 3D spheroid formation of biological ceils of claim 1 , wherein the biological cells are benign tumor ceils.

1 8. The platform for inducing 3 D spheroid formation of biological ceils of claim 1 , wherein the biological cells are malignant tumor cells.

19. The plaiform for inducing 3D spheroid formation of biological cells of claim 1 , wherein the biological ceils are cancer ceils.

20. The platform for inducing 3D spheroid formation of biological ceils of claim 1 , wherein the biological cells are breast cancer ceils.

2 1 . The platform for inducing 3D spheroid formation of biological ceils of claim 1 , wherein the spheroids have diameters of about 10 μηι to about 1000 μπι.

22. The plaiform for inducing 3D spheroid formation of biological ceils of claim 1 , wherein the spheroids have diameters of about 20 μτη to about 200 μπι.

23. A method for inducing 3D spheroid formation of biological ceils on a

thermogeliing polymer without amino acids, comprising the step of: combining the

thermogeliing polymer with a growth medium aod the biological cells.

24. The method of claim 23, wherein the thermogeliing polymer is a stearate- modified methyl cellulose.

25. The method of claim 23, wherein the thennogeliing polymer is poioxamer 407 linked by polyurethane linkages.

26. The method of claim 25., wherein the polyurethane l inkages are generated utilizing hexamethyidiisocyanate.

27. The method of claim 25, wherein the polyurethane linkages are generated util izing methylene diphenyldiisocyanate.

28. The method of claim 23, wherein the polyurethane linkages are generated utilizing toluene diisocyanaie,

29. The method of claim 23, wherein the thermogeliing polymer is a hydrophobicaliy- modified cellulose derivative.

30. The method of claim 23, wherein the ihermoge!fing polymer is a cellulose derivative modified by chemical conjugation of stearic acid units,

3 1 . The method of claim 23, wherein the thermogelling polymer is a cellulose derivative modified by chemical conjugation of methyl units.

32. The method of claim 23, wherein the thermogelling polymer is a cellulose derivative modified by chemical conjugation of hydroxypropyl units.

33. The method of claim 23, wherein the thermogelling polymer is is a cellulose derivative modified by chemical conjugation of ethyl units.

34. The method of claim 23, wherein the thermogelling polymer is a cel lulose derivative modified by chemical conjugation of propyl units.

35. The method of claim 23, wherein the thermogelling polymer is a cellulose derivative modified by chemical conjugation of hydroxyethyi units.

.

36. The method of claim 23, wherein the thermogelling polymer is a cellul ose derivative modified by chemical conjugation of carboxymethyl units.

37. The method of claim 23, wherein the ceil growth medium is in the range of about

1 to about 30% w/v.

38. The method of claim 23, wherein the ceil growth medium is in the range of about 5 to about 20% w/v.

39. The method of claim 23, wherein the biological cells are tumor cells.

40. The method of claim 23, wherein the biological cells are benign tumor ceils.

41. The method of claim 23, wherein the biological cells are malignant tumor ceils.

42. The method of claim 23, wherein the biological cells are cancer cells.

43. The method of claim 23, wherein the biological cells are breast cancer cells.

44. The method of claim 23, wherein the spheroids have diameters of about 10 μτη to about 1 000 μΐΏ.

45. The method of claim 23, wherein the spheroids have diameters of about 20 μηι to about 200 μπι.

46. The method of claim 23, further comprising the step of: incubating the biological cells with die themio gel ling polymer and growth medium at appropriate conditions to support biological growth.

47. The method of claim 46, further comprising the step of: harvesting the 3D spheroid formations of biological ceils from the thermogelling polymer and growth medium.

48. The method of claim 23, further comprising the step of: harvesting the 3D spheroid formations of biological cel ls from the thermogelling polymer and growth medium.